Melanin the Biopolymer
Melanogenesis, production of the biological polymer melanin, is a widespread phenomena in nature occurring in most phyla from fungi to mammals. Tyrosinase (E.C. 1.14.18.1), is known to catalyze melanin formation and is present in bacteria, fungi, gymnosperms, angiosperms, arthropods and chordates. The black, brown, buff and Tyndall-blue pigments found in feathers, hairs, eyes, insect cuticle, fruit and seeds are usually melanins and are assumed to result from the action of tyrosinase. The enzyme is not universal; it occurs relatively rarely in prokaryotes, is absent in a variety of higher plants and is generally confined to specific cells of the skin in higher animals but may occur in interior tissue, such as the substantia nigra, eye and inner ear. Melanins have been assigned a photoprotective role in the skin, their role in the eye and inner ear in unknown.
The mammalian melanins are subdivided into two chemical classes; eumelanins (the brown to black pigments derived from 3,4-dihydroxyphenylalanine [dopa] oxidation products) and pheomelanins (the red to yellow pigments derived from cysteinyldopa oxidation products). The intractable nature of these pigments has made their characterization and quantification experimentally difficult. In humans, eumelanins and pheomelanins exist as an intimate mixture, the ratio of eumelanin to pheomelanin being genetically determined.
The stepwise biosynthesis of melanins is presented in FIG. 1. The first two steps of eu- and pheomelanogenesis are catalyzed by the tyrosine hydroxylase (TH) and dopa oxidase (DO) activities of tyrosinase. Both eu- and pheomelanogenesis proceed by the same pathway to dopaquinone. In the absence of sulfhydryl compounds, eumelanin results. Dopaquinone and its subsequent cyclized intermediates may form eumelanin through a series of nonenzymatic steps. The reactions distal to dopaquinone proceed spontaneously at room temperature and were originally thought to be unregulated. The rate constants, k, for some of these post-dopaquinone reactions enroute to eumelanin have been reported.
______________________________________ Rate constant, k ______________________________________ dopaquinone cyclodopa 134 s.sup.-1 (pH 5.4) dopaquinone + dapachrome + 10.sup.9 s.sup.-1 M.sup.-1 (pH 7.7) cyclodopa dopa dopachrome 5,6-dihydroxy- 5.8 .times. 10.sup.-5 s.sup.-1 (pH 5.1) indole ______________________________________
Chemical regulation of melanogenesis is assumed to be the result of general acid-base catalysis or electrostatic catalysis by nucleophiles and electrophiles inherent to the reaction medium. More notably, the changes in the ionic strength of the reaction medium regulate catalysis by influencing the polarizability of the melanogenic precursors and intermediates. The reaction medium itself may cause solvent-solute interactions which influence permanent or induced dipoles. Regulation is also manifested when changes in pH change the degree of ionization of reactants or the medium. Finally, molecular interactions such as hydrogen bonding, dimerization or ion pair formation among the reactants or medium may regulate melanogenesis.
The following list of "melanin facts" must be kept in mind when trying to define, characterize, or quantify melanin polymers:
1. Melanogenesis not only affords melanins, but also a number of "melanochromes" such as 5,6-dihydroxyindoles, cysteinyldopas and trichochromes. PA1 2. Melanins are more properly referred to as melanoprotein, and are composed of a protein fraction intimately bound to a chromophore. PA1 3. Melanins are known to exist in nature as particles, and it has been demonstrated that the isolation protocol can irreversibly change the nature of the melanin granule. PA1 4. Alkaline peroxide treatment of melanin yields "solubilized" melanin termed melanin free acid (MFA). PA1 5. When heated above 100.degree. C. melanin readily gives off carbon dioxide (this process has been assigned to decarboxylation of aryl carboxylic acid residues present in the polymer). PA1 6. Melanin exhibits ion exchange properties which have been postulated to have importance for the biological function of the pigment. PA1 A. " . . . melansosomes were collected from HP melanoma and secondly the melanin was washed extensively with tetrahydrofuran (THF) to extract impurities. In this step, THF was coloured to yellow. The sample was then dried and was dissolved in alkaline solution, e.g., ethylene diamine water solution (30/100 volume ratio) and/or 1N NH.sub.4 OH water solution. A large volume of 12N HCl water solution was poured into this melanin solution which was then boiled for 30 hours and remained to rest. The precipitated melanin was collected, washed repeatedly, and dialyzed and dried. Finally melanin was washed by THF repeatedly until THF became colourless and then dried." From "Chemico-Physical properties of Melanin (II)." PA1 B. " . . . The black precipitate, collected by centrifugation, was kept in conc HCl at room temp for 7 days. After centrifugation, the melanin was thoroughly washed with 1% HCl distilled water and finally acetone." From "The Structure of Melanins and Melanogenesis-II." PA1 C. "The eyes were dissected to separate the iris, ciliary body, choroid and retinal pigment epithelium. These fractions were pooled and suspended in distilled water and then homogenized. The homogenate was filtered through four layers of gauze and the filtrate was mixed with an equal volume of concentrated HCl to give a final concentration of 6N HCl. The mixture was stirred for 24 hours; the precipitate was removed by centrifugation, resuspended in 6N HCl, and refluxed for 48 hours. The precipitate was washed with water 4-6 times and suspended in water." From "Do the Melanins from Blue and Brown Human Eyes Differ?" Menon, I. A., et al. Pigment Cell 1981, Proceedings of the XI International Pigment Cell Conference Sendai, Japan, pp. 17-22 (1981).
The literature is replete with reports concerning the physical, chemical and biological properties of melanins which have been isolated from animal and plant systems. However, the isolation techniques reported have been poorly designed. They are often chemically harsh and rarely take into consideration the inherent reactivity of melanins. The following three examples depict protocols which are commonly referenced in the literature:
The hypothetical structure of melanin depicted below, incorporates the work of numerous groups over the last five decades. For reviews, see Swan, G. A. Fortschritte of Chem. Org. Naturst. XXXI, 552; (1974); Proto, G. Medical Research Reviews 8, 525 (1988); and Ito, S. Biochim. Biophys. Acta 883, 155 (1986).
The study of melanins has led to the discovery of a number of pathways of biosynthesis and also to a wide variety of chemicals related to melanins. Fungal melanins occur as wall-bound melanins and extracellular melanins. Most hyphal, conidial, and sclerotial walls of melanized fungi appear to have two distinct layers: an inner layer which is electron translucent and an outer layer containing electron-dense granules. Collective evidence shows that these granules are melanins, Wheeler, M. H. et al., Exp. Mycol. 3, 340 (1979). Extracellular melanins are synthesized apart from cell walls. They are derived from phenols by two mechanisms: (a) oxidation of phenolic compounds by phenol oxidases (sometimes also called phenyloxidase) secreted into the medium and (b) oxidation of phenols secreted into the medium either by autooxidation or by enzymes released during autolysis. Wheeler, M. H. et al., Can J. Microbiol. 24, 289 (1978).
Fungi or bacteria which secrete tyrosinase cause discoloration of the surrounding medium. That discoloration can be accentuated by adding tyrosine to the medium, Hollis, J. P., Phytopathology 42,273 (1952); Nurudeen, T. A. et al., J. Clin. Microbiol. 10, 724 (1979). Extracellular melanins have been observed in Actinomycetes, bacteria, and fungi. Genes controlling extracellular tyrosinase production or secretion occur on plasmids in Streptomyces scabies, Gregory, K. F. et al., J. Bacteriol. 87, 1287 (1964), and Rhizobium phaseoli strain 1233, Beynon, J. L. et al., J. Gen. Microbiol. 120, 421 (1980). The tyrosinase gene in Vibrio cholerae is located on the chromosome. Bell, A. A. et al., Ann. Rev. Phytopathol. 24, 411 (1986).
In one of the melanin pathways, synthesis of Eumelanin is mediated by tyrosinase which is generally agreed to catalyze the first two steps in the biosynthesis. The initial reaction involves the hydroxylation of tyrosine. An oxygen atom is incorporated adjacent to the hydroxyl group of tyrosine to produce 3,4-dihydroxyphenylalanine (DOPA). Tyrosinase then catalyses the conversion of DOPA to dopaquinone. The dopaquinone formed is not stable at physiological pH. The amino group of the side chain cyclizes to give cyclodopa which then oxidizes rapidly to dopachrome, a red compound. The next step is a rearrangement and decarboxylation to give 5,6-dihydroxyindole (DHI) or without decarboxylation to produce 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The eumelanins are formed from the polymerization of dopaquinone, dopachrome, DHI and DHICA or combinations thereof. These form the brown pigments in animals. Mason, H. S., J. Biol. Chem. 172, 83 (1948); and Pawelek, J. M. et al., Am. Sci. 70, 136 (1982). Crippa et al., The Alkaloids 36, 253 (1989), Academic Press N.Y., N.Y.
Phaeomelanins, the red, brown and yellow pigments of animals are polymers of cysteinylDOPAs mixed which are derived from mixed cystein and tyrosine. Fitzpatrick, T. B. et al., in Biology and Diseases of Dermal Pigmentation p. 3, Univ. Tokyo Press, Tokyo. Trichochromes are also classified with melanins since they are yellow, red and violet pigments and they are derived from the oxidation of tyrosine.
Allomelanins which contain little or no nitrogen are formed from phenolic precursors, primarily catechol and 1,8 dihydroxynaphthalene.
Tyrosinase is not the only melanin producing enzyme. Laccase, an enzyme found in the outer walls of fungi is responsible for the oxidation of DOPA. Laccase will not readily oxidize tyrosine. Simon, L. T. et al., J. Bacteriol. 137, 537 (1979). Other enzymes present in pigment producing organisms are phenyloxidase of Cryptococcus neoformans as well as catechol oxidase and other polyphenol oxidases of plants. Mayer, A. M. et al., Phytochem. 18,193 (1979).
.gamma.-glutaminyl-3,4-hydroxybenzene (GDHB) melanin is synthesized from .gamma.-glutaminyl-4-hydroxybenzene (GHB) by that action of tyrosinase in Agaricus bisporus. Hegnauer, H. et al., Exp. Mycol. 9, 221. Ustilago maydis is believed to metabolize catechol to melanins. Teleospores of U. maydis produce highly election dense melanins when fixed with OsO.sub.4. Patgieter, H. J. et al., J. Bacteriol. 91, 1526 (1966).
Biosynthesis of 1,8-dihydroxynaphthalene (DHN) melanin is produced from pentaketide. A variety of intermediates occur including 1,3,6,8-tetra-hydroxynaphathlene, scytalone, 1,3,8-trihydroxyl-naphthalene, vermelone, dihydroxynaphthalene, dihydroxynaphthalene 1,1-dimer and dihydroxynaphthalene 2,2-dimer. Mutational blocks eliminating reductase or dehydratase enzymes, and enzyme inhibitors such as tricyclazole cause the occurrence of a large number of shunt products. Wheeler, M. H. et al., Arch. Microbiol. 142, 234 (1985); and Stipanovic, R. D. et al., Pestic. Biochem. Physiol. 13, 198 (1980).
Culture conditions vary among different microorganisms. Production of Extracellular melanins in some microorganisms has been shown to increase as the concentration of tyrosine is increased to its saturation point of 0.1 percent. This percentage is considered supersaturation in tyrosine. Hollis, J. P. (1952), supra. It has been reported that yeast autolysates and casein hydrolysate stimulate melanin pigment production by Streptomyces scabies in a medium containing 0.1% percent tyrosine. Hollis, J. P. (1952), supra.
It has also been shown that production of melanins is repressed by a variety of carbon sources. The particular carben source varies with the microorganism. Nurudeen, T. A. et al. (1979), supra, reported that increased glucose concentration in the medium reduced pigmentation of all serotypes of Cryptococcus neoformans. This fungus produces melanin-like pigments with diphenol and aminophenol through the mediation of a phenyloxidase enzyme. The phenyloxidase of C. neoformans cannot use tyrosine as a substrate. In contrast to the metabolism of Cryptococcus, Gluconobacter oxydans, a pigment producing bacterium produces melanin in the presence of glucose and tyrosine, but not in a medium containing sucrose, fructose, sorbitol, mannitol or glycerol as the carbon source. Pavlenko, G. V. et al., Microbiology USSR 50, 539 (1981).
Several fungi are known to produce extracellular heterogenous melanins. These melanins are derived from various phenols, amino acids, proteins, carbohydrates and lipids. Synthesis requires secretion of tyrosinase into the medium.
Many species of Streptomyces are capable of forming dark melanin pigments due to expression of tyrosinase from the mel gene locus. The mel locus of S. antibioticus has been cloned and sequenced, Katz, E. et al., J. Gen. Microbiol. 123, 2703 (1983); Bernan, V. et al., Gene 37, 101 (1985) and shown to contain two open reading frames (ORF's) that encode a putatuve ORF438 protein (M.sub.r =14,754) and tyrosinase (M.sub.r =30,612). ORF438 and tyrosinase are thought to be transcribed from the same promoter in S. antibioticus, and both genes are required for melanin production. Bernan, V. et al., (1985), Supra. Based on genetic evidence, ORF438 protein has been shown to function as a trans-activator of tyrosinase. Lee, Y. -H. W. et al., Gene 65, 71 (1988). It has been suggested that the ORF438 protein is involved in tyrosinase secretion, or it may function as a metallothionein-like protein that delivers copper to apotyrosinase, Bernan, V. et al., (1985), Supra; Lee, Y. -H. W. et al., (1988), Supra. The mel locus of S. glaucescens has a nearly identical ORF sequence upstream of tyrosinase that probably serves a similar function. Huber, M. et al., Biochemistry 24 6038 (1985); Huber, M. et al., Nucleic Acids Res. 15 8106 (1987). The existence of an ORF438 protein, however, has never been confirmed in vivo.
Naturally occurring E. coil does not have a tyrosinase gene and does not produce melanin. The BclI fragment of plasmid pIJ703 encoding the tyrosinase gene of Streptomyces lividans was cloned into plasmid YEp13 at the BamHI site and transformed into E. coli HB101. There was no detectible expression of tyrosinase or expression of melanin. Nayak, K. et al., Indian Journal of Biochemistry & Biophysics 25, 515 (1988).
U.S. Pat. No. 4,898,814 issued to Kwon discloses a cDNA clone of human tyrosinase and claims a method of making human tyrosinase by expressing the cDNA in E. coli.
Melanin production in Shewanella colwelliana, a gram negative marine bacterium, has been analyzed by measuring L-DOPA synthesis in crude extracts. The region encoding melanin syntheses was mapped and sequenced. A pair of open reading frames (ORF) were found. One ORF was found to correspond to the tyrosinase gene. The downstream ORF encoded a polypeptide of unknown function. Deletion of the downstream ORF was found to have no effect on pigmentation in E. coli transformed with the tyrosinase gene. Fuqua, W. et al., Abstract, American Society for Microbiology Washington, D.C. Branch of George Mason University (1990).